Advance Journal of Food Science and Technology 5(11): 1443-1449, 2013
ISSN: 2042-4868; e-ISSN: 2042-4876
© Maxwell Scientific Organization, 2013
Submitted: June 28, 2013
Accepted: July 08, 2013
Published: November 05, 2013
Effects of Synthetic and Natural Extraction Chemicals on Functional Properties,
Polyphenol Content and Antioxidant Activity of Soy Protein Isolates
Extracted from Full-Fat and Defatted Flours
1, 2
Moses Vernonxious Madalitso Chamba, 1Yufei Hua, 1Quirino Dawa,
1
Odilon Djakpo and 1Caimeng Zhang
1
School of Food Science and Technology, Jiangnan University, 1800 Lihu
Avenue, Wuxi 214122, Jiangsu Province, P.R. China
2
Department of Human Ecology, Domasi College of Education, University of Malawi,
P.O. Box 49, Domasi, Zomba, Malawi
Abstract: The number of consumers preferring natural and organic foods to those that involve synthetic chemicals
has recently shown dramatic growth. As such, a native isoelectrically precipitated soy protein isolates (SPIs) were
prepared from full-fat and defatted flours using amaranth (Amaranthus tricolor L.) lye (pH>12.5) and lemon extract
(pH<2.5) as natural food-plant-based chemicals, replacing the conventional synthetic ones (NaOH and HCl,
respectively). Functional properties, total polyphenol content and antioxidant activity of the natural SPIs were
compared to those of synthetic ones. All the SPI samples qualified to be soy protein isolates with a minimum protein
content of 91.21%. The natural SPI showed significant increase in emulsion stability (p≤0.05). While higher values
with narrow margins were shown by the synthetic than the natural SPIs in oil absorption (0.66±0.02, 0.50±0.01%,
respectively), emulsion capacity (56.53±0.57, 55.50±0.39%), foam stability (11.33±0.61, 10.40±0.40%). No
significant difference was observed in water absorption capacity. The DPPH assay showed increased antioxidant
activity in the natural SPI although its total polyphenol content was lower. Thus, SPI with functional properties
similar to those of conventional ones can be naturally processed using amaranth ash and lemon extracts as
alternative chemicals to addressing the fear of consuming synthetic chemical by health-conscious natural and
organic food consumers.
Keywords: Amaranth ash extract, conventional soy protein isolate, lemon extract, natural chemicals, natural soy
protein isolate, synthetic chemicals
INTRODUCTION
The past few years have shown dramatic growth in
organic, natural and environmentally friendly or
"green" food market. As many people are becoming
more conscious about their health, uncertainty about
safety of the highly processed foods, especially in terms
of its long term effect as they interact with one another
in the body, is rising. Studies on food preferences have
shown fear of exposure to non-food synthetic chemicals
and environmental consciousness as major factors
influencing this new food consumer behavior (DicksonSpillmann et al., 2011). Recent years have witnessed an
attempt by the agricultural industry to produce food
naturally without using synthetic chemicals. Likewise,
food manufacturers are also trying to explore new food
processing techniques to address the need for safer food
and compete for consumer acceptance (Zink, 1997).
Various modern and partially traditional techniques
are currently being employed by the food industry to
process plant agricultural products, such as soybeans,
into fat-reduced, protein-enriched compositions for use
in food manufacturing. They include solvent extraction
and a variety of press-based methods, e.g., extruder,
expeller, continuous and cold presses, to separate at
least a portion of the fat from the remaining plant
material. Nevertheless, at some stage strong non-food
synthetic chemicals are still used to finalize the process.
Some of these chemicals are not natural, or plant based
and cannot be used to produce certified organic food
products under United States Department of Agriculture
(USDA) guidelines for organic food labeling (Gold,
2012). As such, a lot of improvements are yet to be
made on highly processed foods.
Soy protein isolate (SPI), a soybean derivative
protein powder, is one of the most important products
in food processing as well as many other industrial
uses. Its preference is attributed to its ability to
enhance nutritional (especially protein) and functional
qualities of food products to which it is used as an
ingredient (Kinsella, 1979; Mariotti et al., 1999;
L’hocine et al., 2006). SPI has been added to baked
foods, breakfast cereals and meat products among
others. It can also be made into a nutritious drink once
Corresponding Author: Yufei Hua, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi
214122, Jiangsu Province, P.R. China
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Adv. J. Food Sci. Technol., 5(11): 1443-1449, 2013
added to water. A soybean product qualifies to be called
a protein isolate only if it contains at least 90% crude
protein (N×6.25) on dry basis (Codex Alimentarius
Commission, 1996). To achieve this purity,
conventional technique of processing SPI involves use
of synthetic chemicals such as n-hexane, ethanol,
sodium hydroxide (NaOH) and hydrochloric acid (HCl)
(Kinsella, 1979). These chemicals have well known
risks if handled without taking adequate precautions
and their health hazard cases, especially with direct
contact in adequate doses have been widely reported.
Although some of these chemicals are removed during
the process, having some small residues in the final
product cannot be exclusively denied. Furthermore, a
health conscious natural food consumer cannot be
convinced that foods processed using these chemicals
are safe to eat. Dickson-Spillmann et al. (2011)
reported that laypeople perceive chemicals as either
safe or hazardous and think that even minor doses of
chemicals are likely to cause harm. To such people,
‘synthetic equals dangerous’. The challenge lies
therefore on identifying natural and food-based reagents
and chemicals that would produce similar or improved
results as the synthetic ones. At this time, when
consumers are increasingly becoming skeptical about
consuming food processed using synthetic chemicals,
more exploration on the traditional chemistry may be
considered a favorable option just as it is in traditional
medicine.
Amaranth ash solution has been used traditionally
for food preparation as an alternative to bicarbonate of
soda (alkaline). In countries like Malawi, amaranth
(Amaranthus hybridus) plant is the most preferred
source of ash for food alkali due to its pH strength and
safety for consumption. However, information on the
same can scarcely be found. Lemon extract as citric
acid is a well known food acid, but rarely mentioned in
soybean products studies.
In our previous study it was observed that soy
protein isolate with acceptable yield, composition and
nutritive quality can be produced using purely natural,
food-plant-based chemicals such as amaranth ash and
lemon extracts as alternative to NaOH and HCl,
respectively (Chamba et al., 2013). The aim of this
study was therefore; to examine the functional
properties (water and oil absorption capacity,
emulsifying capacity and stability, foam formation and
stability, gelation), total polyphenol content (TPC) and
antioxidant activity of the SPIs prepared using these
natural chemicals with comparison to those in which
the conventional synthetic chemicals were involved.
purchased from a local market. Soybean grains were
supplied by Suzhou Golden Village Food Co., Ltd.
Jiangsu province, China. The soybeans were sorted,
cleaned (to remove unrelated materials), dehulled then
ground into native full fat flour, fine enough to pass
through an 80 mesh sieve. On dry basis, the flour had
35% protein (N×6.25), 24% lipid, 3% ash, 7% moisture
and Protein dispersibility index (PDI) of 72%. One part
of the flour was treated with n-hexane and 95% alcohol
to remove fat and soluble sugars. Amaranth ash extract
(pH≥12.50) was prepared by filtering distilled water
through burnt amaranth plant ash at the ash to water
ratio of 1:5(w/v) after optimization. Lemon extract
(pH<2.20) was prepared by squeezing the lemons then
vacuum filtering the extract to remove the pulp. DPPH
(2, 2-diphenyl-1-picryl hydrazyl) was purchased from
Sigma, Shanghai. All other reagents and chemicals
were of analytical grade. Deionized or distilled water
was used in all laboratory procedures and sample
preparations.
Preparation of soy protein isolate: Four SPI samples
namely; Synthetic Chemical Full Fat flour (SCFF),
Synthetic Chemical Defatted Flour (SCDF) Natural
Chemical Full Fat flour (NCFF) and Natural Chemical
Defatted Flour (NCDF) were prepared as described by
Chamba et al. (2013). Appropriate flour (full fat or
defatted) was suspended in distilled water at the flour to
water ratio of 1:10 (w/v). The pH of the suspension was
adjusted to and maintained at 7.0 with either amaranth
extract or NaOH, while stirred for 1 h at ambient
temperature 22±2°C. After centrifugation, at 10000×g
and 4°C for 30 min (for SCFF, SCDF and NCDF SPIs),
the supernatant was recovered. Soy protein was
precipitated by adjusting pH to 4.5 with lemon extract
or HCl then centrifuged at the above conditions. The
precipitate was washed and centrifuged twice before
suspended again in distilled water and neutralized to pH
7.0 with either amaranth extract or NaOH. All
resolubilized SPI samples were freeze-dried, sealed in
polythene bags and stored at 4°C until further analysis.
METHODOLOGY
Proximate analyses: Proximate analyses of the four
samples were conducted as follows: Crude protein was
determined by micro-Kjeldahl method with the
common conversion factor of N×6.25 (AOAC, 2000).
Crude oil was extracted the traditional Soxhlet
extraction apparatus and determined according to
AOAC official method (AOAC, 2000). Total ash was
analyzed using the conventional method by dry-ashing
in muffle furnace at 550°C. Moisture content was
calculated by drying weighed samples for 3 h in an
oven at 120°C (Smith et al., 1966).
Materials: Edible green and purple colored amaranth
(Amaranthus tricolor L.) plants were obtained from
local farmers in the outskirt of Wuxi city, Jiangsu
province, China. Ripe lemon (Citrus limon) fruits were
Functional properties:
Oil and water absorption capacities: Oil absorption
capacity (OAC) and Water Absorption Capacity
(WAC) were assessed in triplicate using the procedure
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described by Lin et al. (1974). A protein sample (0.3 g)
was mixed with soy oil or distilled water (1.5 mL) in a
pre-weighed 15 mL graduated centrifuge tubes and
centrifuged for 30 min at 4000 rpm. The supernatant
was discarded and the tubes were reweighed. The OAC
and WAC (%) were expressed using the following
equation:
FAC/WAC (%) = (W 0 + W 1 ) /W 0 ×100
where,
W 0 = The initial weight of the sample
W 1 = The weight of sample plus the absorbed liquid
Foaming capacity and stability: Foams were prepared
according to Sathe and Salunkhe (1981) with minor
modifications. Briefly, 2 g of each SPI sample was
solubilised in 200 mL of distilled water to make a 1%
solution. Aliquots of 50 mL were poured into graduated
250 mL, 3 cm diameter cylinder and whipped by high
shear stepless speed homogenizer (model: FA 25, Fluco
Equipment Co. Ltd., Shanghai, China) at 10000 rpm,
ambient temperature (20±2°C) for 3 min. A smooth
round end glass rod was used to level the foam for easy
reading. Foam capacity was calculated as percentage
rise in total volume at 0 times. Stability was determined
as a factor of foam drainage recorded after 30 min:
Foaming capacity (%) = (V 1 - V 0 ) /V 0 ×100
Foam stability (%) = V 2 /V 0 ×100
where,
V 0 = Initial sample volume before whipping
V 1 = Total sample volume after whipping at time 0
V 2 = The water volume still trapped in foam after 30
min
Emulsifying capacity and stability: For emulsion
properties, the method described by Yasumatsu et al.
(1972) was used with minor modification. Protein
isolate solution (1%, w/v) was prepared in 30 mL of
distilled water in a beaker, magnetically stirred for 1 h
to solubilize the protein. Then, 30 mL of soy oil was
added. The mixture was homogenized by a rod
homogeniser (model: FA 25, Fluco equipment co. Ltd.,
Shanghai, China) for 0.5 min at 10000 rpm. Triplicate
aliquots (12 mL each) of the freshly prepared emulsion
was poured into calibrated centrifuge tubes and
centrifuged at 2500 rpm for 15 min. The ratio of the
height of water layer to the total liquid layer was used
to calculate emulsifying activity (EA). The emulsion
was then heated at 70°C for 30 min in a water bath,
followed by 15 min of cooling under running tap water
and centrifugation at the same conditions. The stability
was worked out using the height of the two layers.
Results were calculated as follows:
EA/ES (%) = (V 0 - V 1 ) /V 0 ×100
where,
V 0 = The initial volume of the total liquid in the
centrifuge tube
V 1 = The volume of water layer
Least gelation concentration: The least gelation
concentration was determined by the method of
Coffman and Garcia (1977). Test tubes containing
5 mL protein suspensions of 5, 6, 7, 8 up to 15%,
respectively (w/v) in distilled water were heated for 1 h
in boiling water, followed by cooling in ice bath and
further cooling for 2 h at 4°C. The results were reported
as liquefied (-), gluey (±) and gel (+). The least gelation
concentration was the minimum concentration at which
the sample did not fall down or slip when the test tube
was inverted.
Determination of total polyphenol content and
antioxidant activities:
Total polyphenol contents (TPC): To extract
polyphenols, a sample (1 g) was suspended in 25 mL
methanol-water 80:20 v/v acidified with 0.1% HCl and
magnetically stirred for 1 h at room temperature. Later,
the mixture was centrifuged at 1800×g for 15 min, the
methanol was decanted and the residue extracted again
with 25 mL of fresh aqueous methanol. After
centrifugation at same conditions, the two extracts were
combined (Akowuah et al., 2005; Mujica et al., 2009).
Total phenols content (TPC) of the SPIs was
determined by Folin-Ciocalteau phenol reagent as
described by Škerget et al. (2005) with some
modification, using garlic acid monohydrate (0-60
µg/L, N = 7) as a standard. To 0.5 mL of appropriately
diluted sample, 2.5 mL of Folin-Ciocalteu reagent was
added. After that (within time interval from 0.5 to 8
min), 2 mL of Na 2 CO 3 (7.5%) was added. The mixture
was incubated for 5 min at 50°C and then cooled. For a
control sample, 0.5 mL of distilled water instead of
sample was used. The absorbance was measured at 760
nm by UV/Vis spectrophotometer (UV-2450, Shimadzu
Corporation, Japan). The content of total phenols was
expressed as garlic acid equivalents (GAE) in µg/g SPI.
All analyses were run in triplicate and mean values
were calculated. The calibration equation of garlic acid
was y = 0.05997x - 0.03535, R2 = 0.9997.
DPPH free radical-scavenging assay: DPPH free
radical-scavenging assay was determined according to
the method of Brand-Williams et al. (1995) with
modification. A 2 g of each SPI sample was extracted
twice (2 h each time) with 20 mL aqueous methanol
(80% containing 0.1% HCl) centrifuged at 2500 rpm
for 15 min and supernatant combined. Then, a series of
working solutions of different concentrations (0.5, 1, 2,
3, 4 and 5 mg/mL, respectively) were prepared. A
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Adv. J. Food Sci. Technol., 5(11): 1443-1449, 2013
Table 1: Proximate analyses of the SPI samples
Content
NCFF
NCDF
Protein (%)
91.21
93.64
Lipid
0.71
<0.05
Ash
5.42
5.91
Moisture
3.38
3.24
SCFF
92.12
0.65
3.83
2.49
SCDF
98.45
<0.05
2.08
2.84
3.5 mL of methanolic DPPH (2, 2-diphenyl-1-picryl
hydrazyl, 0.06 mM) solution was added to 1.5 mL
diluted sample or methanol (for control) and vortexed.
The mixture was incubated for 30 min at room
temperature in the dark. The absorbance was read at
517 nm by UV-Vis spectrophotometer (UV-2450,
Shimadzu Corporation, Japan) against the DPPH
solvent as blank. The inhibition percentage of free
radical by sample was calculated using the following
formula and the results presented as a comparative
graph:
Inhibition % = (A 0 - A 1 ) /A 0 ×100
where,
A 0 = The absorbance of the control (blank, without
sample)
A 1 = The absorbance in the presence of the sample
Statistical analysis: One way analysis of variance
(ANOVA), with Duncan's multiple range test, was
conducted using a SAS program (version 8.1, SAS
Institute Inc., Cary, NC, USA) to asses significance of
differences (p<0.05) among means of triplicate sample
runs.
RESULTS AND DISCUSSION
Proximate analysis: Values for proximate analysis are
presented in Table 1. The SPIs prepared from either
full-fat soybean flour or natural chemicals (ash and
lemon extracts) showed slightly lower protein
compositions as compared to the one from both defatted
flour and conventional synthetic chemicals (NaOH and
HCl), respectively.
A soy product is recognized as an isolate only if it
contains not less than 90% protein (Preeti et al., 2008).
Thus all samples, including the NCFF, qualified to be
SPIs. The rest of the compositions were within the
recommended ranges of a good quality SPI according
CODEX STAN 175-1989 general standards (Codex
Alimentarius Commission, 1996). The lower protein
composition observed in NCFF may be attributed to
high fat in the starting material (full-fat flour), ash
composition in the solvent (ash extract) and remaining
pulp in the acid (lemon extract).
Functional properties:
Oil and water absorption capacities: Results of the
oil and water absorption capacities are presented in
Table 2. The WAC was significantly higher in SCFF
SPI (0.62±0.05 mL/g) followed by SCDF and NCFF
(0.55±0.04, 0.55±0.03 mL/g, respectively). NCDF SPI
showed the lowest value. In their study, Fleming et al.
(1974) and Deshpande et al. (1982) attributed improved
water absorption capacity to the increase in protein
content of the sample. Similarly, higher WAC has been
observed in protein isolates in comparison with protein
concentrates (Fleming et al., 1974; Lin et al., 1974;
Wang and Kinsella, 1976; Hutton and Campbell, 1977)
probably indicating the relationship between water
absorption and protein content. However, the findings
of this study did not fully agree with the protein content
of the samples (Table 1). Inasmuch as protein content
between the two main samples (SCDF and NCFF) was
substantially different, there was no difference in water
absorption, indicating that other factors might had
played a role. Lin et al. (1974) observed that sunflower
products had lower water absorptions than did soy
products, although their protein contents were similar.
These finding indicate that water absorption of a
particular sample need not always be parallel to the
protein content; other food components may have an
influence.
The oil absorption capacity (OAC) differed
significantly
in
all
samples
(SCDF>
SCFF>NCFF>NCDF) as shown in Table 2. The
differences were within a range between 0.37±0.01
mL/g (NCDF) and 0.66±0.02 mL/g (SCDF) for all
samples and from 0.50±0.01 mL/g (NCFF) to
0.66±0.02 mL/g (SCDF) between the two main samples
(traditional and conventional SPIs, NCFF and SCDF,
respectively). Oil absorption values seemed to fall
within the same ranges with those of water absorption.
Oil absorption is mainly attributed to the physical
entrapment of oil and is related to the number of
nonpolar side chains on proteins that bind hydrocarbon
chains of fat (Lin et al., 1974; Kinsella, 1979). The
increased absorption in SCDF is then associated with
increased nonpolar side chains in the sample due to its
higher protein content.
Foaming capacity and stability: Volume increase of
the 1% aqueous dispersion of all the SPI samples
ranged from 110.00±5.29% (NCFF) to 231.33±4.16%
(SCDF) after whipping as shown in Table 2. The
specific volumes of the foams were determines as
indicators of air uptake during whipping. The lower air
Table 2: Water and oil absorption, emulsion and foaming properties of the traditional and conventional soy protein isolates
Sample
Water absorption (mL/g) Oil absorption (mL/g) Emulsion capacity (%) Emulsion stability (%)
Forming capacity (%)
SCFF
0.62±0.05a
0.59±0.01b
55.83±0.66b
52.30±1.64b
127.33±3.06c
a
a
b
b
SCDF
0.55±0.04
0.66±0.02
56.53±0.57
54.51±0.92
231.33±4.16a
NCFF
0.55±0.03a
0.50±0.01c
55.50±0.39c
54.71±1.00a
110.00±5.29d
NCDF
0.32±0.05b
0.37±0.01d
57.47±0.33a
53.43±0.89b
217.33±7.02b
a-d
: Values with different superscript letters along the same column indicate significant difference (p≤0.05) among means of triplicates tests
1446
Form stability (%)
9.80±0.70b
11.33±0.61a
10.40±0.40b
9.60±0.40b
Adv. J. Food Sci. Technol., 5(11): 1443-1449, 2013
uptake of the SPI prepared with the natural chemicals
and full-fat flour was associated with decrease in its
foam volume. Specific volume of foam (volume per
unit mass) for a given set of conditions is an indication
of air uptake only and is not necessarily a measure of
foam quality and stability (Deshpande et al., 1982).
The foam stability was determined as a factor of
foam drainage after 30 min, other than the volume
maintained by of the foam. Observation has shown that
foam has a capability of maintaining their volume by
sticking to the side of the container even if it is weak.
Usually, the amount of liquid retained in the foam is a
better indication of foam strength or quality. Although
the total volumes just after whipping at ambient
temperature (16±1°C) differed significantly (p<0.05) in
all samples (Table 2), their stabilities did not differ
except in SCDF, where it was higher. Thus the stability
of the foam may not only be attributed to protein
concentration alone as indicated by Deshpande et al.
(1982), but also to other components to which the liquid
gets entrapped. The effects of pH on both foam capacity
and stability have been reported (Lin et al., 1974; Sekul
et al., 1978; Canella et al., 1979; Cherry and
McWatters, 1981). However, the fact that all samples
were treated under the same pH condition, it may not be
associated with the differences.
Emulsifying capacity and stability: The emulsion
capacity (EC) and emulsion stability (ES) of all the SPI
samples are given in Table 2. Comparison of the main
natural and conventional samples (NCFF and SCDF)
showed a significant decrease (p<0.05) of EC with a
very narrow margin (1.86%) in NCFF than SCDF SPIs.
The highest EC was observed in one of the intermediate
samples (NCDF). However, the natural SPI
demonstrated the most significantly (p<0.05) stable
emulsion than the rest of the samples, whose ES did not
differ. At the same concentration, the ES of all the
samples seemed lower that those reported by others.
This may be attributed to the method used to prepare
the emulsions. It has been reported that in emulsions
prepared using the shearing force method, the
stabilization property of heated soy protein isolate
decreased to a lower level when it was compared to
those prepared by other methods (Zayas, 1997).
Stabilization of the emulsions by protein polymers is
relevant in almost all food applications. Protein
polymers have two effects: They can gel in the
continuous phase at high protein concentrations through
protein cross-linkings and they provide strong repulsive
forces when absorbed at the oil/water interface (Ma and
Barbosa-Cánovas, 1995).
Least gelation concentration: Gelation may be
defined as a protein aggregation phenomenon in which
polymers-polymer and polymer-solvent interactions and
attractive and repulsion forces are so balanced that a
tertiary network or matrix is formed (Schmidt, 1981).
Such matrix has a capability of trapping and
immobilising large amount of water. Protein
Table 3: Least gelation concentration of SPI samplesa
Concentration (%) SCFF
SCDF
NCFF
NCDF
5
(-)
(-)
(-)
(-)
6
(-)
(-)
(-)
(-)
7
(-)
(-)
(-)
(-)
8
(±)
(-)
(-)
(-)
9
(±)
(±)
(-)
(±)
10
(±)
(+)
(±)
(+)
11
(+)
(+)
(±)
(+)
12
(+)
(+)
(±)
(+)
13
(+)
(+)
(±)
(+)
14
(+)
(+)
(+)
(+)
15
(+)
(+)
(+)
(+)
LGC
11
10
14
10
a
: Gelation levels: (-) liquefied, (±) gluey and (+) gel; The values are
the mean of three replicates
concentration, other protein components, in a complex
food system, non protein components, heat treatment
conditions, pH and ionic and reducing agents are some
of the factors that affect gelation (Schmidt, 1981).
Comparison of the main natural and conventional SPIs
indicated similarity with the findings observed in the
foaming property, where higher values were observed
in the SPIs prepared from defatted flour than full-fat
flour. The greatest value was recorded in NCFF (14%)
and the lowest in SCDF and NCDF (both 10%) as
presented in Table 3. This was not surprising as it
related to the protein content of the samples, which was
higher in those prepared from defatted soy flour and
was least in NCFF SPI.
Depending on food product to which a protein
isolate is used as an ingredient, both high and low
gelation properties are desirable. These results showed
that the natural chemical prepared SPI had lower
gelation property compared to the conventional one. By
using non-defatted flour and lemon and ash extracts,
which were not thoroughly purified, NCFF contained a
complex of particles and compound such as
carbohydrates and metal elements that might interfere
with formation of continuous network of the protein
molecules to form a gel. A higher protein concentration
thus may be required to overcome their interference
with gel formation. Schmidt et al. (1978) reported that
dialysis of whey protein concentrate system improved
its gelation property. Nevertheless, more information is
required on the effects of components other than the
SPI itself on protein gelation properties before
predictions regarding the gelation behavior of this
natural SPI in a complex food system can be made.
Determination of total polyphenol content and
antioxidant activities:
Total polyphenol contents (TPC): The results of the
Total polyphenol content (TPC) showed a wide
difference between samples prepared using different
chemicals, but no significant differences were observed
in SPIs prepared from the same chemicals. SCFF and
SCDF contained higher TPC (23.80±1.93 and
22.69±2.67 µg GAE/g, respectively) than both NCFF
(11.02±1.27 µg GAE/g) and NCDF (9.40±1.33 µg
GAE/g). Consequently, it was expected that the
conventional chemically prepared samples would
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Adv. J. Food Sci. Technol., 5(11): 1443-1449, 2013
CONCLUSION
25
NCFF SPI
SCFF SPI
NCDF SPI
SCDF SPI
Percentage inhibition
20
15
10
5
0
0
2
4
6
8
10
12
14
16
Sample concentration (mg/ml)
Fig. 1: Antioxidant activity by 2, 2-diphenyl-1-picryl
hydrazyl (DPPH, 0.06 mM) assay
demonstrate increased antioxidant activity than their
natural counterparts. Nevertheless, many compounds
such as vitamin C, which is contained in lemons among
other sources, are also responsible for the antioxidant
activity. This had to be verified by conducting an
antioxidant activity determination.
DPPH free radical-scavenging assay: DPPH free
radical-scavenging assay, using either 2, 2-diphenyl-1picryl hydrazyl or 1, 1-diphenyl-2-picryl hydrazyl
radical is one of the most common methods for
evaluating antioxidant activity of food samples. It is
simple, rapid, generally accurate and inexpensive
(Prakash et al., 2001). Additionally, different authors
have used different ways of reporting DPPH
antioxidant assay results either graphically or by
calculating IC 50, a value at which 50% of the used
DPPH has reacted with the sample (Marinova and
Batchvarov, 2011). In this study, the graphical
presentation was preferred to show the exact absorption
behavior of the samples. As shown in Fig. 1, the DPPH
assay results of this study showed that the SPI prepared
using the traditional chemical demonstrated increased
antioxidant activity (with NCFF having the highest)
than those using conventional chemicals. This increase
may be attributed to the presence of lemon extract,
which is one of the well known sources of vitamin C, a
natural antioxidant. If this is really the case, then the
activity may vary depending on the vitamin C
composition of lemons used.
It was also observed that at a certain concentration,
the natural SPI (NCFF) showed serious reduction in
absorbance regardless of repetition of the test for a
number of times. There is possibility that, due to the
impurity of the natural chemicals, the sample contained
certain components such as carotenoids, which are
normally present in the lemons that at increased
concentration substantially interfered with coloration of
the assay (Noruma et al., 1997), but not necessarily the
antioxidant activity of the sample.
The findings of this study suggest that instead of
the conventional synthetic NaOH and HCl, traditional
chemicals such as amaranth plant ash and lemon
extracts can be used to prepare SPI, not only with
desirable functional properties similar to those of the
conventional ones, but also with increased antioxidant
activity. Basic as it may seem, this method of SPI
processing may have effective outcomes of improving
livelihood of indigenous people in terms of food
supplementation, in addition to providing a possible
alternative to the synthetic chemicals, which are
increasingly feared by the health-conscious food
consumers. Much as the functional properties of soy
protein isolates have been associated with the protein
content of the product, the findings of this study also
suggest that functional properties of SPI cannot
apparently be attributes to the protein level, but also to
its interaction with other components such as ash
particles, carbohydrates and lipids.
ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance
of some students and staff of Vegetable Protein
Laboratory of the School of Food Science and
Technology, Jiangnan University, China.
REFERENCES
Akowuah, G.A., Z. Ismail, I. Norhayati and A. Sadikun,
2005. The effects of different extraction solvents of
varying polarities on polyphenols of orthosiphon
stamineus and evaluation of the free radical
scavenging activity. Food Chem., 93: 311-317.
AOAC, 2000. Official Methods of Analysis. 17th Edn.,
Association of Official Analytical Chemists, Gait
Hersburg, Maryland, USA.
Brand-Williams, W., M.E. Cuvelier and C. Berset,
1995. Use of a free radical method to evaluate
antioxi dant activity. LWT-Food Sci. Technol., 28:
25-30.
Canella, M., G. Castriotta and A. Bernadi, 1979.
Functional and physical chemical properties of
succinylated and acetylated sunflower protein.
Lebensm. Wiss. Technol., 12: 95-101.
Chamba, M.V.M., Y. Hua, N. Murekatete and Y. Chen,
2013. Effects of synthetic and natural extraction
chemicals on yield, composition and protein
quality of soy protein isolates extracted from fullfat and defatted flours. J. Food Sci. Technol, DOI
10.1007/s13197-013-1084-x.
Cherry, J.P. and K.H. McWatters, 1981. Whippability
and Aeration. In: Cherry, J.P. (Ed.), Protein
Functionality in Foods. ACS Symposium Series
147, American Chemical Society, Washington DC,
pp: 149.
1448
Adv. J. Food Sci. Technol., 5(11): 1443-1449, 2013
Codex Alimentarius Commission, 1996. Codex
Alimentarius-Cereal, Purses, Legumes and Protein
Derived Products and Vegetable Proteins. Codex
Alimentarius Commission, Rome, Italy.
Coffman, C.W. and V.V. Garcia, 1977. Functional
proper ties and amino acid contents of protein
isolate from mung bean flour. J. Food Technol., 12:
473-484.
Deshpande, S.S., S.K. Sathe, D. Cornforth and
D.K. Salunkhe, 1982. Effect of dehulling of
functional properties of dry bean (Phaseolus
vulgaris L.) Flours. Cereal Chem., 59: 39-401.
Dickson-Spillmann, M., M. Siegrist and C. Keller,
2011. Attitudes toward chemicals are associated
with preference for natural food. Food Qual.
Prefer., 22: 149-156.
Fleming, S.E., F.W. Sosulski, A. Kilara and
E.S. Humbert, 1974. Viscosity and water
absorption characteristics of slurries of sunflower
and soybean flours, concentrates and isolates.
J. Food Sci., 39: 188-193.
Gold, M.V., 2012. Organic Production/Organic Food:
Information Access Tool. IOP USDA Web.
Retrieved from: http://www.nal.usda.gov/afsic/
pubs/ofp/ofp.shtml, (Accessed on: August 10,
2012).
Hutton, W.C. and A.M. Campbell, 1977. Functional
properties of a soy isolate and a soy concentrate in
simple system as related to performance in a food
system. J. Food Sci., 42: 457-460.
Kinsella, J.E., 1979. Functional properties of soy
protein isolate. J. Am. Oil. Chem. Soc., 56:
242-258.
L’hocine, L., J.I. Boye and Y. Arcand, 2006.
Composition and functional properties of soy
protein isolate prepared using alternative defatting
and extraction procedures. J. Food Sci., 71:
C137-C145.
Lin, M.J.Y., E.S. Humbert and F.W. Sosulski, 1974.
Certain functional properties of sunflower meal
products. J. Food Sci., 39: 368-370.
Ma, L. and V. Barbosa-Cánovas, 1995. Rheological
characterization of mayonnaise. Part II: Flow and
viscoelastic properties at different oil and xanthan
gum concentrations. J. Food Eng., 25: 409-425.
Marinova, G. and V. Batchvarov, 2011. Evaluation of
the methods for determination of the free radical
scavenging activity by DPPH. Bulg. J. Agric. Sci.,
17: 11-24.
Mariotti, F., S. Mahé, R. Benamouzig, C. Luengo,
S. Daré, C. Gaudichon and D. Tomé, 1999.
Nutritional value of [15N]-soy protein isolate
assessed from ileal digestibility and postprandial
protein utilization in humans. J. Nutr., 129:
1992-1997.
Mujica, M.V., M. Granito and N. Soto, 2009.
Importance of the extraction method in the
quantification of total phenolic compounds in
Phaseolus vulgaris. L. Interciencia, 34: 650-654.
Noruma, T., M. Kikuchi and Y. Kawakami, 1997.
Proton-donative antioxidant activity of fucoxanthin
with 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH).
Biochem. Mol. Biol. Int., 42: 361-370.
Prakash, A., F. Rigelhof and E. Miller, 2001.
Antioxidant Activity. In: DeVries, J. (Ed.),
Medallion Laboratories Analytical Progress.
Medallion Laboratories, Minnesota, Minneapolis,
19(2): 1-6.
Preeti, S., R. Kumar, S.N. Sabapathy and A.S. Bawa,
2008. Functional and edible uses of soy protein
products. Compr. Rev. Food Sci. Food Safety, 7:
14-28.
Sathe, S.K. and D.K Salunkhe, 1981. Functional
properties of great northern bean (Phaseolus
vulgaris) proteins: Emulsion, foaming, viscosity
and gelation properties. J. Food Sci., 46: 71-75.
Schmidt, R.H., 1981. Gelation and Coagulation. In:
Cherry, J.P. (Ed.), Protein Functionality in Foods.
ACS Symposium Series 147, American Chemical
Society, Washington DC, pp: 131.
Schmidt, R.H., B.L. Illingworth and E.M. Ahmed,
1978. The effect of dialysis on heat-induced
gelation of whey protein concentrate. J. Food
Process. Pres., 2: 111-221.
Sekul, A.A., C.H. Vinnett and R.L. Ory, 1978. Some
functional properties of peanut proteins partially
hydrolysed with papain. J. Agric. Food Chem., 26:
855-858.
Škerget, M., P. Kotnik, M. Hadolin, A.R. Hraš,
M. Simonič and Z. Knez, 2005. Phenols,
proanthocyanidins, flavones and flavonols in some
plant materials and their antioxidant activities.
Food Chem., 89: 191-198.
Smith, A.K., J.J. Rackis, P. Isnardi, J.L. Cartter and
O.A. Krober, 1966. Nitrogen solubility index,
isolated protein yield and whey nitrogen content of
several soybean strains. Cereal Chem., 43:
261-270.
Wang, J.C. and J.E. Kinsella, 1976. Functional
properties of novel proteins: Alfafa leaf protein.
J. Food Sci., 41: 286-292.
Yasumatsu, K., K. Sawada, S. Moritaka, M. Mikasi,
J. Toda, T. Wada and K. Ishii, 1972. Whipping and
emulsifying properties of soybean products. Agric
Biochem., 36: 719-727.
Zayas, J.F., 1997. Functional Property of Protein in
Food. Springer-Verlag, Berlin, Heidelberg,
Germany.
Zink, D.L., 1997. The impact of consumer demands and
trends on food processing. Emerg. Infect. Dis., 3:
467-469.
1449